Such a method of repeatedly removing a surface layer (also known as slicing) and obtaining an image of a specimen is known from e.g. particle-optical apparatus having both an ion-optical column and an electron-optical column, such as e.g. the DualBeam® instruments commercially available from FEI Company.
It is remarked that ‘an image’ in the context of this invention is to be interpreted as an image displayed on a display unit as well as a representation thereof in e.g. a computer memory.
Such a method is used in industry and laboratories, e.g. to analyze and inspect biological specimens and polymeric specimens and e.g. to form three-dimensional (3D) reconstructions of structures in biological tissues and polymers.
The instrument used for performing the known method comprises an electron-optical column to obtain an image of a specimen by scanning a focused beam of energetic electrons, typically with an energy between 0.1 to 30 keV, over the specimen. The working of such a column is known from a Scanning Electron Microscope (SEM). Where the beam of electrons impinges on the specimen, secondary radiation, such as secondary electrons, backscattered electron, X-rays and light, may be emitted in response to the bombardment with the impinging electrons. By detecting the amount of e.g. secondary electrons emitted with e.g. a Secondary Electron Detector (SED), (place dependent) information of the surface of the specimen can be obtained. This information can be displayed as an image on a display, or the image can be stored for future retrieval or processing.
After thus obtaining an image of the surface of the specimen, a surface layer may be removed using the ion column. The working of such a column is known from Focused Ion Beam (FIB) instruments. The column emits a focused beam of energetic ions, such as a beam of Ga+ ions with an energy of e.g. 40 keV. Where the beam of ions impinges on the specimen, material is removed. This removal is greatly enhanced by admitting certain gasses in the vicinity where the beam impinges on the specimen. This ion beam can be scanned over the surface, whereby the dwell time (together with the ion beam properties such as current density and energy) determines how much of the surface layer is removed. As a result a slice of material is removed.
After the removal of the surface layer a fresh surface layer is exposed, and with the electron beam an image can be obtained of the thus exposed surface layer. By repeatedly obtaining an image of a surface layer and removing the surface layer from the specimen (removing a slice from the surface layer), a 3D reconstruction of the specimen can be made. Alternatively, a region of interest in the interior of the specimen can be brought to the surface to be examined by techniques that e.g. offer surface information.
A problem when observing certain materials, such as polymers and biological tissues, is that the contrast of the specimen may be too poor to easily differentiate features of the specimen. As known to the person skilled in the art, in order to improve contrast, specimens may be stained to preferentially highlight some parts of the specimen over others. For stains to be effective, they have to preferentially bind to some parts of the specimen, thereby differentiating between different parts of the specimen.
In electron microscopy, heavy metal salts may be used as a staining agent. Such heavy metal salts are commonly derived from gold, uranium, ruthenium, osmium, or tungsten. Heavy ions are used since they will readily interact with the electron beam and produce phase contrast, absorption contrast and/or cause backscattered electrons. Some of these heavy metal salts adhere to specific substances of the specimen. An example of that is OsO4 (osmiumtetroxide), which form a specific chemical reaction with the —C=C— double bonds of unsaturated fatty acids.
Other staining agents that may be used are e.g. compounds of a heavy metal with e.g. an appropriate biologically active group, such as an antibody. Such staining agents are also known as labels. An example is colloidal gold particles absorbed to antibodies. Other examples of this group of staining agents are the Nanogold® particles, produced by Nanoprobes Inc., USA, which may be used to label any molecule with a suitable reactive group such as oligonucleotides, lipids, peptides, proteins, and enzyme inhibitors.
To stain a specimen the specimen is exposed to the staining agent. The exposure can take the form of temporarily immersing the specimen in a liquid, such as a 1% solution of OsO4. Further steps in the staining process may include washing the specimen with water, alcohol, etc. Such staining processes are e.g. described by “Dermatan sulphate-rich proteoglycan associates with rat-tendon collagen at the d band in the gap region”, John E. Scott and Constance R. Orford, Biochem. J. (1981) 197, pages 213-216, more specific in the section ‘materials and methods’.
The exposure can also take the form of exposing the specimen to a gas or vapour of the staining agent. This is e.g. described in “Observation on backscattered electron image (BEI) of a scanning electron microscope (SEM) in semi-thin sections prepared for light microscopy”, Y. Nagata et al., Tokai J. Exp. Clin. Med., 1983 May 8(2), pages 167-174.
For a good contrast the specimen must be sufficiently stained. There is however an optimum in the staining dose: too much staining results in a decrease of the contrast as too much of the specimen becomes stained, whereby the (stained) structures of interest do not stand out to the background anymore. An adequate dose of staining must thus be found.
A problem with certain combinations of staining agents and the materials to be stained is that the diffusion rate of the staining agent in the specimen is very low. Many of the heavy metal staining agents show a low diffusion rate in biological tissues, while in polymers the diffusion rate is even lower. As a result, when e.g. thick polymeric specimens are stained such that the surface is stained to an adequate level, the interior of the specimen is insufficiently stained to obtain a good contrast. If however the staining is such, that the interior is sufficiently stained, the surface is so heavily stained as to be unfit for obtaining a good image. There is therefore a need to stain thick specimens in such a way, that the whole specimen is stained to an adequate level.
The invention intends to provide a method for staining thick specimens in such a way, that the whole specimen can be imaged with an adequate staining level. To that end the method according to the invention is characterized in that, after at least one of the removals of a surface layer the specimen is exposed to a staining agent. By re-staining the surface of the specimen every time that a surface layer is stripped, the surface can be stained to the optimum level as well as a constant level every time.
It is remarked that it might be that re-staining is not necessary after every individual removal of a surface layer, but only after a predetermined number of layers. This may lead to a reduced processing time and thus shorter cycle time.